Hibernating mammals exhibit unparalleled tolerance to a degree of cerebral ischemia that is debilitating or fatal to non-hibernators, including humans. The molecular mechanisms underlying natural neuroprotection during hibernation are largely unknown. Three recent advances: 1) the discovery of adaptive RNA editing of ion channels in cold vs warm-living octopus; 2) a growing appreciation of post-transcriptional mechanisms that alter the protein output of the transcriptome; and 3) data revealing few quantitative differences in the brain proteome of hibernators, lead us to propose that temperature-sensitive RNA editing is retained as an adaptation for heterothermy in mammalian hibernators. Specifically, we hypothesize that temperature-sensitive RNA editing by Adenosine Deaminase Acting on RNA (ADAR) alters protein structure and function in hibernator brains to enhance neuroprotection. ADAR activity substitutes adenosine with inosine (A-to-I) in double-stranded RNA; the double-stranded character of RNA is increasingly stabilized as temperature is decreased. A-to-I replacements can re-code amino acids and alter splice acceptor, splice donor, or 3' end processing and polyadenylation sites to significantly alter the protein output of the transcriptome. Brain proteins including ion channels and neuroreceptors are important targets of ADAR editing in several species, including mammals. We will study three brain regions with dramatically different activities across hibernation stages, exploiting our unique ground squirrel tissue bank which contains 200 samples from precisely defined, distinct physiological states, and includes animals with core body temperatures ranging from 5C to 37C. Aim One will characterize the transcriptome from multiple stages of hibernation that differ by body temperature using RNA- seq. Data will be analyzed to identify transcripts undergoing apparent state-specific RNA editing that would cause amino acid substitution or splice variants impacting protein structure. Putative RNA editing changes will be validated by comparing conventional sequencing chromatograms from cDNA and genomic DNA PCR products. A second, more specific round of sequencing experiments in Aim Two will use a targeted approach to selectively enrich detection of transcripts with alternative 3' ends. At the end of thi exploratory/developmental project we will have a greatly enhanced understanding of how the brain transcriptome alters across phenotypic cycles of the hibernator's year. If RNA editing is detected in hibernator brain, we also will have determined whether it increases as body temperature decreases in a mammal. By identifying specific targets of RNA editing and other structural variation, these experiments may reveal protein isoforms underlying the hibernator's enhanced neuroprotection and provide a critical first step towards engineering an analogous neuroprotected stated in victims of cerebral ischemia by stroke, cardiac arrest or trauma.